Evaluation of Clinically Significant miRNAs Level by Machine Learning Approaches Utilizing Total Transcriptome Data.

Analysis of the mechanisms underlying the occurrence and progression of cancer represents a key objective in contemporary clinical bioinformatics and molecular biology. Utilizing omics data, particularly transcriptomes, enables a detailed characterization of expression patterns and post-transcriptional regulation across various RNA types relative to the entire transcriptome. Here, we assembled a dataset comprising transcriptomic data from approximately 16 000 patients encompassing over 160 types of cancer. We employed state-of-the-art gradient boosting algorithms to discern intricate correlations in the expression levels of four clinically significant microRNAs, specifically, hsa-mir-21, hsa-let-7a-1, hsa-let-7b, and hsa-let-7i, with the expression levels of the remaining 60 660 unique RNAs. Our analysis revealed a dependence of the expression levels of the studied microRNAs on the concentrations of several small nucleolar RNAs and regulatory long noncoding RNAs. Notably, the roles of these RNAs in the development of specific cancer types had been previously established through experimental evidence. Subsequent evaluation of the created database will facilitate the identification of a broader spectrum of overarching dependencies related to changes in the expression levels of various RNA classes in diverse cancers. In future, it will make possible to discover unique alterations specific to certain types of malignant transformations.

Antigenic Peptide-Thioredoxin Fusion Chimeras for In Vitro Stimulus of CD4+ TCR+ Jurkat T Cells.

Study of CD4+ T cell response and T cell receptor (TCR) specificity is crucial for understanding etiology of immune-mediated diseases and developing targeted therapies. However, solubility, accessibility, and stability of synthetic antigenic peptides used in T cell assays may be a critical point in such studies. Here we present a T cell activation reporter system using recombinant proteins containing antigenic epitopes fused with bacterial thioredoxin (trx-peptides) and obtained by bacterial expression. We report that co-incubation of CD4+ HA1.7 TCR+ reporter Jurkat 76 TRP cells with CD80+ HLA-DRB1*01:01+ HeLa cells or CD4+ Ob.1A12 TCR+ Jurkat 76 TRP with CD80+ HLA-DRB1*15:01+ HeLa cells resulted in activation of reporter Jurkat 76 TPR after addition of recombinant trx-peptide fusion proteins, containing TCR-specific epitopes. Trx-peptides were comparable with corresponding synthetic peptides in their capacity to activate Jurkat 76 TPR. These data demonstrate that thioredoxin as a carrier protein (trx) for antigenic peptides exhibits minimal interference with recognition of MHC-specific peptides by TCRs and consequent T cell activation. Our findings highlight potential feasibility of trx-peptides as a reagent for assessing the immunogenicity of antigenic fragments.

[NETosis in lupus nephritis].

AIM: To evaluate the levels of MPO-DNA complex in patients with systemic lupus erythematosus (SLE) and its association with the presence of lupus nephritis (LN). MATERIALS AND METHODS: The study included 77 patients with SLE, of whom 30 had SLE without anti phospholipid syndrome (APS), 47 had SLE with APS, and 20 were healthy individuals serving as the control group. The MPO-DNA complex in the serum was investigated using ELISA. RESULTS: The levels of MPO-DNA complex in serum were significantly higher in patients with SLE compared to healthy controls (p=0.001). Among the patients with SLE, 30 (39%) had elevated levels of MPO-DNA complex. The presence of elevated MPO-DNA complex was significantly associated with the presence of a history of LN (p=0.009). Moreover, among the patients included in the study, 20 had active LN, and patients with elevated MPO-DNA complex levels were more likely to have active LN than patients without elevated MPO-DNA complex concentrations [12 (40%) of 30 vs 8 (17%) of 47, χ2=5.029; p=0.034]. An association was found between elevated levels of MPO-DNA complex and the presence of proteinuria, hematuria, cellular hematic/granular casts and aseptic leukocyturia. A direct correlation of MPO-DNA complex with SLEDAI-R was found in patients with active LN (rs=0.497; p=0.026). CONCLUSION: Elevated levels of MPO-DNA complex were detected in 39% of patients with SLE. These patients had a higher prevalence of LN in their medical history and at the time of inclusion in the study. The correlation between MPO-DNA complex levels and the activity of LN according to SLEDAI-R indicates the potential role of MPO-DNA complex as a biomarker for assessing the activity of renal damage in SLE.

Barnase-barstar Specific Interaction Regulates Car-T Cells Cytotoxic Activity toward Malignancy.

The development of CAR-T specific therapy made a revolution in modern oncology. Despite the pronounced therapeutic effects, this novel approach displayed several crucial limitations caused by the complications in pharmacokinetics and pharmacodynamics controls. The presence of the several severe medical complications of CAR-T therapy initiated a set of attempts aimed to regulate their activity in vivo. We propose to apply the barnase-barstar system to control the cytotoxic antitumor activity of CAR-T cells. To menage the regulation targeting effect of the system we propose to use barstar-modified CAR-T cells together with barnase-based molecules. Barnase was fused with designed ankyrin repeat proteins (DARPins) specific to tumor antigens HER2 (human epidermal growth factor receptor 2) The application of the system demonstrates the pronounced regulatory effects of CAR-T targeting.

A new mouse unilateral model of diffuse alveolar damage of the lung.

OBJECTIVE AND DESIGN: The existing biological models of diffuse alveolar damage (DAD) in mice have many shortcomings. To offset these shortcomings, we have proposed a simple, nonsurgical, and reproducible method of unilateral total damage of the left lung in ICR mice. This model is based on the intrabronchial administration of a mixture of bacterial lipopolysaccharide (LPS) from the cell wall of S. enterica and α-galactosylceramide (inducing substances) to the left lung. METHODS: Using computer tomography of the lungs with endobronchial administration of contrast material, we have been able to perform an operative intravital verification of the targeted delivery of the inducer. The model presented is characterized by more serious and homogeneous damage of the affected lung compared to the existing models of focal pneumonia; at the same time, our model is characterized by longer animal survival since the right lung remains intact. RESULTS: The model is also characterized by diffuse alveolar damage of the left lung, animal survival of 100%, abrupt increases in plasma levels of TNFa, INFg, and IL-6, and significant myocardial overload in the right heart. It can be used to assess the efficacy of innovative drugs for the treatment of DAD and ARDS as the clinical manifestations that are developed in patients infected with SARS-CoV-2. Morphological patterns of lungs in the noninfectious ("sterile") model of DAD induced by LPS simultaneously with α-galactosylceramide (presented here) and in the infectious model of DAD induced by SARS-CoV-2 have been compared. CONCLUSION: The DAD model we have proposed can be widely used for studying the efficacy of candidate molecules for the treatment of infectious respiratory diseases, such as viral pneumonias of different etiology, including SARS-CoV-2.

Topological Features of Histone H2A Monoubiquitination.

The cellular response to DNA damage protects the essential information stored in the genome. This mechanism is crucial in terms of the cancer prevention and aging progression. The DNA damage response (DDR) consists of a complex network controlling the cell cycle and multiple mechanisms of the DNA repair. The DDR disruption is a cornerstone feature of the tumor cells, which allows them to enhance beneficial mutations that prevent successful disease treatment. The important checkpoints of the DDR are currently poorly understood due to the complexity and diversity of the DNA repair machinery. Histone ubiquitination is intensively involved in the repair of the double-stranded DNA breaks. This post-translational modification is known to be a key factor in the recruitment of the repair factors to the DNA damage sites. Here, the crucial role of the ubiquitin lysine residue K27 in the process of histone H2A monoubiquitination mediated by the ubiquitin ligase RNF168 has been showed. The presented data suggest forced and intensive diffusion of ubiquitin from the cytoplasm to the nucleus, which is characterized by the dynamic equilibrium less than 10 min. The comparison of the turnover rate of the wild-type ubiquitin and its variant with a single functional lysine residue K27 suggests an important role of the ubiquitin deposition as a covalent conjugate with histone H2A in terms of the stability of the entire ubiquitinome.

[Pharmacoinvasive Approach to the Treatment of Acute ST-Segment Elevation Myocardial Infarction. Current State of the Problem].

The articled focused on the pharmacoinvasive approach to the treatment of acute ST-segment elevation myocardial infarction. Current guidelines prioritize the primary transcutaneous coronary intervention. However, in both the Russian Federation and other countries, there are some peculiarities (logistic issues, specific aspects of infrastructure of medical facilities), which may hamper timely conduction of the endovascular treatment. In such cases, the thrombolytic therapy subsequently supplemented with transcutaneous coronary intervention would appear the most effective strategy aimed at the earliest recovery of coronary perfusion. The authors provided results of major studies that used such approach and the effect of using the thrombolytic therapy with recombinant prourokinase, a Russian third generation thrombolytic drug.

[Non-coding RNAs as therapeutic targets in spinal cord injury]. / Nekodiruyushchie RNK kak terapevticheskie misheni pri travme spinnogo mozga.

Spinal cord injury (SCI) may be followed by persistent motor dysfunction and somatosensory disturbances that negatively influences the quality of life of patients and creates a significant economic burden. Analysis of secondary biological processes associated with changes in genetic expression is becoming increasingly important every day in understanding the pathophysiology of spinal cord injury. The results of international sequencing of the human genome were analyzed in 2004. These data revealed about 20,000 protein-coding genes covering near 2% of the total genomic sequence. The vast majority of gene transcripts are actually characterized as non-coding RNAs (ncRNAs). These RNA clusters do not encode functional proteins and ensure post-transcriptional regulation of gene expression. The clusters may be small (approximately 20 nucleotides) known as miRNAs or the transcripts can enroll over 200 nucleotides defined as long non-coding RNAs (lncRNAs). Some modern studies describe transient expression of microRNA in case of spinal cord injury. These RNAs are associated with inflammation and apoptosis, functional recovery and regeneration. Large-scale genomic analysis has demonstrated the existence of multiple lncRNAs whose expression is associated with some processes of spinal cord injury. lncRNA can be divided into two categories depending on the position in relation to the coding genes: intergenic and intragenic. Intergenic lncRNAs is currently the most studied class. Intragenic lncRNAs can be subdivided depending on the overlap of the coding genes (antisense, intron, etc.). According to recent studies, long non-coding RNAs are abundantly present in the tissues of central nervous system and may be crucial in the pathogenesis of certain diseases of nervous system. At the cellular level, it has been shown that lncRNAs regulate the expression of protein-coding RNAs. Moreover, these molecules are involved into such processes as neuronal death, demyelination and glia activation. This review is devoted to the role of ncRNAs in the pathogenesis of spinal cord injury and their potential use as targets for the treatment of consequences of spinal cord injury.

Proteasome: a Nanomachinery of Creative Destruction.

In the middle of the 20th century, it was postulated that degradation of intracellular proteins is a stochastic process. More than fifty years of intense studies have finally proven that protein degradation is a very complex and tightly regulated in time and space process that plays an incredibly important role in the vast majority of metabolic pathways. Degradation of more than a half of intracellular proteins is controlled by a hierarchically aligned and evolutionarily perfect system consisting of many components, the main ones being ubiquitin ligases and proteasomes, together referred to as the ubiquitin-proteasome system (UPS). The UPS includes more than 1000 individual components, and most of them are critical for the cell functioning and survival. In addition to the well-known signaling functions of ubiquitination, such as modification of substrates for proteasomal degradation and DNA repair, polyubiquitin (polyUb) chains are involved in other important cellular processes, e.g., cell cycle regulation, immunity, protein degradation in mitochondria, and even mRNA stability. This incredible variety of ubiquitination functions is related to the ubiquitin ability to form branching chains through the ε-amino group of any of seven lysine residues in its sequence. Deubiquitination is accomplished by proteins of the deubiquitinating enzyme family. The second main component of the UPS is proteasome, a multisubunit proteinase complex that, in addition to the degradation of functionally exhausted and damaged proteins, regulates many important cellular processes through controlled degradation of substrates, for example, transcription factors and cyclins. In addition to the ubiquitin-dependent-mediated degradation, there is also ubiquitin-independent degradation, when the proteolytic signal is either an intrinsic protein sequence or shuttle molecule. Protein hydrolysis is a critically important cellular function; therefore, any abnormalities in this process lead to systemic impairments further transforming into serious diseases, such as diabetes, malignant transformation, and neurodegenerative disorders (multiple sclerosis, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease and Huntington's disease). In this review, we discuss the mechanisms that orchestrate all components of the UPS, as well as the plurality of the fine-tuning pathways of proteasomal degradation.

High-Throughput Platform for B-Cell Screening Based on Fluorescent Phage-Display Technology.

A system for detection of malignantly transformed cells, including follicular lymphoma Bcells, was developed and experimentally validated. The system is based on the use of bacteriophages carrying exposed ligands for pathogenic B-cell receptors. The efficiency of binding to target cells is several times higher than in systems with chemically synthesized biotinylated peptides. The new method is proposed as a noninvasive diagnostic test for mapping B-cell lymphoma and for determining the specificity of B-cell receptors and high-throughput combinatorial selection of various repertories of B cells.

Differential Diagnostics of Active Progressing Multiple Sclerosis Using a Fluorescent Biomarker with Resonance Energy Transfer.

Previous data showed that myelin-reactive autoantibodies found in patients with multiple sclerosis and mice with experimental autoimmune encephalomyelitis recognize and hydrolyze various fragments of myelin basic protein (MBP). Moreover, antibody-mediated cleavage of the encephalithogenic fragment MBP81-103 flanked with two fluorescent proteins can serve as a new biomarker of multiple sclerosis. Here we describe creation of the next generation of this biomarker based on antibody-dependent degradation of a new chemically synthesized fluorescent substrate with resonance energy transfer that contains fluorophore Cy5 and quencher QXL680 separated by MBP81-99 protein (Cy5-MBP81-99-QXL680). This substrate is degraded during incubation with purified antibodies and B cells from patients with multiple sclerosis, but not healthy volunteers.

Loading Rate of Exogenous and Autoantigenic Determinants on Major Histocompatibility Complex Class II Mediates Resistance to Multiple Sclerosis.

Genetic analysis of thousands of patients with multiple sclerosis (MS) and healthy Russian donors showed that the carriage of groups of HLA-DRB1*15 and HLA-DRB1*03 alleles is associated with the risk of MS, whereas the carriage of groups of HLA-DRB1*01 and HLA-DRB1*11 alleles is protective. Recombinant HLA-DRB1*01:01 with a high affinity can recognize the fragments of myelin basic protein (MBP), one of the autoantigens in MS. However, the comparison of the kinetic parameters of the load of MBP and viral HA peptides on HLA-DRB1*01:01, which is catalyzed by HLA-DM, showed a significantly lower rate of exchange of CLIP for MBP peptides. We assume that the observed protective properties of the group of HLA-DRB1*01 alleles may be directly associated with the ability of HLA-DRB1*01:01 to kinetically distinguish peptides of exogenous and endogenous nature.

Combinatorial Screening of Peptides, Specific Ligands of Death Receptor DR5.

Death receptors, in particular DR5, are highly attractive targets of antitumor therapy. The major limitation to application of natural death receptor ligands (TRAIL) is their non-specific cytotoxicity against normal cells. Since TRAIL can also bind decoy receptors (DcR) and prevent induction of apoptosis, the search for new DR-specific ligands is a topical issue. In the present study, we used combinatorial phage display peptide libraries to select a panel of DR5-binding amino acid sequences. A comparative analysis of the selected peptides enabled identification of the consensus sequence responsible for binding to DR5. Integration of this motif into polypeptide cytotoxic agents may provide targeted elimination of malignantly transformed cells.

Design of Chemical Conjugate for Targeted Therapy of Multiple Sclerosis Based of Constant Fragment of Human Antibody Heavy Chain and Peptoid Analog of Autoantigen MOG35-55.

Elimination of B cells producing autoantibodies to neuroantigens is considered as beneficial in the treatment of multiple sclerosis. Myelin oligodendrocyte glycoprotein (MOG) is a significant autoantigen in multiple sclerosis. It was shown that MOG-like peptoid AMogP3 can bind autoantibodies produced by pathological lymphocytes. We propose a structure of an innovative drug for targeted elimination of the pool of autoreactive B cells responsible for multiple sclerosis pathogenesis; this compound is a complex of peptoid AMogP3 with Fc fragment of human immunoglobulin. The obtained Fc-PEG-AMogP3 conjugate effectively interact with autoreactive antibodies, which attests to their high therapeutic potential.

Analysis of Immunogenicity of Intracellular CTAR Fragments of Epstein-Barr Virus Latent Phase Protein LMP1.

Intracellular fragments of latent phase protein LMP1 of Epstein-Barr virus, denoted as CTAR1/2/3, can trigger a variety of cell cascades and contribute to the transforming potential of the virus. Generation of recombinant proteins CTAR1/2/3 is expected to yield more ample data on functional and immunogenic characteristics of LMP1. We created genetic constructs for prokaryotic expression of LMP1 CTAR fragments and selected optimal conditions for their production and purification. Using a new library of LMP1 CTAR fragments, we carried out epitope mapping of a diagnostic anti-LMP1 antibody S12. Analysis of polyclonal serum antibodies from mice immunized with full-length LMP1 confirmed immunogenicity of CTAR elements comparable with that of full-length protein.

Evolution of catalytic centers of antibodies by virtual screening of broad repertoire of mutants using supercomputer.

It is proposed to perform quantum mechanical/molecular dynamics calculations of chemical reactions that are planned to be catalyzed by antibodies and then conduct a virtual screening of the library of potential antibody mutants to select an optimal biocatalyst. We tested the effectiveness of this approach by the example of hydrolysis of organophosphorus toxicant paraoxon using kinetic approaches and X-ray analysis of the antibody biocatalyst designed de novo.

Mediators and Biomarkers of Inflammation in Meningitis: Cytokine and Peptidome Profiling of Cerebrospinal Fluid.

Differential diagnosis of bacterial and viral meningitis is an urgent problem of the modern clinical medicine. Early and accurate detection of meningitis etiology largely determines the strategy of its treatment and significantly increases the likelihood of a favorable outcome for the patient. In the present work, we analyzed the peptidome and cytokine profiles of cerebrospinal fluid (CSF) of 17 patients with meningitis of bacterial and viral etiology and of 20 neurologically healthy controls. In addition to the identified peptides (potential biomarkers), we found significant differences in the cytokine status of the CSF of the patients. We found that cut-off of 100 pg/ml of IL-1ß, TNF, and GM-CSF levels discriminates bacterial and viral meningitis with 100% specificity and selectivity. We demonstrated for the first time the reduction in the level of two cytokines, IL-13 and GM-CSF, in the CSF of patients with viral meningitis in comparison with the controls. The decrease in GM-CSF level in the CSF of patients with viral meningitis can be explained by a disproportionate increase in the levels of cytokines IL-10, IFN-γ, and IL-4, which inhibit the GM-CSF expression, whereas IL-1, IL-6, and TNF activate it. These observations suggest an additional approach for differential diagnosis of bacterial and viral meningitis based on the normalized ratio IL-10/IL-1ß and IL-10/TNF > 1, as well as on the ratio IFN-γ/IL-1ß and IFN-γ/TNF < 0.1. Our findings extend the panel of promising clinical and diagnostic biomarkers of viral and bacterial meningitis and reveal opposite changes in the cytokine expression in meningitis due to compensatory action of pro- and antiinflammatory factors.

Peptidyl Aldehyde Specifically Interacts with Immunosubunit ß1i Proteasome: In Vitro and In Vivo Effects.

We studied the effect of ß1i-specific peptidyl aldehyde IPSI-001 on proteasome from mammalian cells. In concentrations <1 µM, this agent effectively suppressed immunoproteasome, but only slightly reduced chymotrypsin-like activity of constitutive proteasome. Intraperitoneal administration of this inhibitor to C3H/He mice in a dose of 100 mg/kg induced no significant physiological or behavioral changes, which attested to its considerable therapeutic potential in the treatment of autoimmune neurodegenerative pathologies.

Heterodimer HLA-DM Fused with Constant Fragment of the Heavy Chain of the Human Immunoglobulin Accelerates Influenza Hemagglutinin HA306-318 Loading to HLA-DR1.

Major histocompatibility complex class II (MHC II) plays an important role not only in the adaptive immune responses to foreign pathogens, but also in the development of some autoimmune diseases. Non-classical MHC, HLA-DM is directly involved in MHC II loading with the peptide. To study this process, we synthesized recombinant proteins HLA-DR1 and HLA-DM. α/ß-Chains of DR1 heterodimer contained C-terminal leucine domains of the fos and jun factors, respectively. Each DM chain contained constant fragment of human antibody heavy chain fused via a long linker domain. In addition, DM α-chain carried N165D substitution suppressing potential glycosylation at this site. We observed significant acceleration of DR1 peptide loading with influenza HA306-318 hemagglutinin in the presence of DM, which indicates functionality of recombinant DR1-DM protein couple. Our results can be used to study the presentation of other viral and self-antigens and can become the basis for the development of new drug modeling.

Deimination of the myelin basic protein decelerates its proteasome-mediated metabolism.

Deimination of myelin basic protein (MBP) by peptidylarginine deiminase (PAD) prevents its binding to the proteasome and decelerates its degradation by the proteasome in mammalian cells. Potential anticancer drug tetrazole analogue of chloramidine 2, at concentrations greater than 1 µM inhibits the enzymatic activity of PAD in vitro. The observed acceleration of proteasome hydrolysis of MBP to antigenic peptides in the presence of PAD inhibitor may increase the efficiency of lesion of the central nervous system by cytotoxic lymphocytes in multiple sclerosis. We therefore suggest that clinical trials and the introduction of PAD inhibitors in clinical practice for the treatment of malignant neoplasms should be performed only after a careful analysis of their potential effect on the induction of autoimmune neurodegeneration processes.